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Molecular Endocrinology, doi:10.1210/me.2005-0110
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Molecular Endocrinology 20 (3): 544-559
Copyright © 2006 by The Endocrine Society

Multifunction Steroid Receptor Coactivator, E6-Associated Protein, Is Involved in Development of the Prostate Gland

Obaid Y. Khan1, Guilian Fu1, Ayesha Ismail, Sathish Srinivasan, Xuni Cao, Yaping Tu, Shan Lu and Zafar Nawaz

Department of Biochemistry and Molecular Biology (O.Y.K., G.F., A.I., S.S., Z.N.), University of Miami Miller School of Medicine, Miami, Florida 33136; Department of Pharmacology (X.C., Y.T.), Creighton University, Omaha, Nebraska 68178; and Department of Pathology and Laboratory Medicine (S.L.), University of Cincinnati College of Medicine, Cincinnati, Ohio 45267

Address all correspondence and requests for reprints to: Dr. Zafar Nawaz, Department of Biochemistry and Molecular Biology, Braman Breast Cancer Institute (M-877), University of Miami Miller School of Medicine, Batchelor’s Building, Room 416, 1580 Northwest 10 Avenue, Miami, Florida 33136. E-mail: znawaz{at}med.miami.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this study we report that deletion of E6-associated protein (E6-AP) in mice results in a smaller prostate gland compared with that in normal wild-type animals. To investigate the mechanism(s) by which E6-AP affects prostate gland growth and development, we carried out both in vitro and in vivo experiments. In this study we show that E6-AP interacts with androgen receptor (AR) in a hormone-dependent manner and enhances the transactivation function of AR. Our in vivo data from E6-AP-null prostate glands show that the level of AR protein is elevated while the level of the AR target protein, probasin, is decreased. In contrast, the level of AR protein is decreased, and its target protein is increased in an E6-AP-overexpressing stable cell line, suggesting that E6-AP modulates both the protein level and the activity of AR. In addition, we show that the levels of phosphatidylinositol 3-kinase, total Akt, and phosphorylated Akt are decreased in E6-AP-null prostate, suggesting that E6-AP deletion down-regulates the signaling of the phosphatidylinositol 3-kinase-Akt pathway. We also show that RhoA negatively regulates AR function, and RhoA levels are increased in E6-AP-null prostate. Furthermore, expression levels of p53, Bax, active caspases, and apoptotic index are increased in E6-AP-null prostate. Collectively, our data suggest that E6-AP deletion attenuates the growth and development of the prostate gland by interfering with AR function as well as by stimulating p53-mediated apoptosis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE MALE STEROID hormone, androgen, influences the development of the prostate gland through its intracellular receptor protein, androgen receptor (AR) (1). In the absence of hormone, AR is associated with cellular chaperones and is localized in the cytoplasm of target cells. Upon binding to hormone, the AR undergoes a series of well-defined steps that include conformational change, dissociation from cellular chaperones, receptor dimerization, phosphorylation, and translocation to nucleus. In the nucleus, AR interacts with coactivators and chromatin-modifying enzymes and subsequently recruits general transcription factors to form a stable preinitiation complex, culminating in the induction of target gene transcription (2).

The steroid hormone receptor coactivators (SRCs) represent a growing class of proteins that interact with receptors in a ligand-specific manner and serve to enhance their transcriptional activity (3, 4, 5, 6). Coactivators have been shown to possess enzymatic activities, such as histone acetyltransferase (7), histone methyltransferase (8), ubiquitin-conjugation (9), and ubiquitin-protein ligase (10). Presumably, the coactivator’s in vivo functions manifest by congregating their enzymatic activities to the promoter region of the target gene (11, 12). Due to their ability to enhance receptor-mediated gene expression, coactivators are considered to play an important role in regulating the magnitude of the biological response to steroid hormones. The level of coactivator expression determines the activity of the receptor in target tissues and the varied hormone response seen among individuals within a population.

We have previously reported the isolation and functional characterization of an E3 ubiquitin-protein ligase, E6-associated protein (E6-AP), as a novel dual-function SRC (10). E6-AP not only enhances hormone-dependent transcriptional activities of various steroid hormone receptors, but also is a member of the E3 class of functionally related ubiquitin-protein ligases. E3 ligases have been proposed to play a major role in defining the substrate specificity of the ubiquitin system (10, 13). Protein ubiquitination involves two other classes of enzymes, namely the E1 ubiquitin-activating enzyme (UBA) and E2 ubiquitin-conjugating enzymes (UBCs). UBA first activates ubiquitin in an ATP-dependent manner, and the activated ubiquitin then forms a thioester bond between the carboxyl-terminal glycine residue of ubiquitin and a cysteine residue of the UBA. Next, ubiquitin is transferred from the E1 to one of the several E2s (UBCs), preserving the high-energy thioester bond. In some cases, ubiquitin is transferred directly from E2 to the target protein through an isopeptide bond between the {epsilon}-amino group of lysine residues of the target protein and the carboxy terminus of ubiquitin. In other instances, the transfer of ubiquitin from UBCs to target proteins proceeds through an E3 ubiquitin-protein ligase intermediate, such as E6-AP. Finally, the ubiquitin-tagged target proteins undergo degradation via the 26S proteasome pathway (14).

In the normal prostate, the processes of cell survival, proliferation, and differentiation are regulated to a large extent by androgens through their interaction with AR (1, 15). Normal prostate secretory cells require physiological levels of androgens for their maintenance, and after castration, the rat prostate rapidly involutes as a result of a major loss of cells (60–70% within 7 d of androgen deprivation) (15, 16). Conversely, sustained androgen administration to mature castrated rats can elicit growth of the prostate gland, which can return to its original size (17). Furthermore, AR-null male mice show an ambiguous or feminized appearance, with the penis appearing as a microphallus with poorly developed scrotum, whereas the vas deferens, epididymis, seminal vesicle, and prostate fail to develop (18). These observations indicate a clear involvement of the AR in the development and maintenance of a normal prostate gland. In addition to androgen signaling, the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway is another important factor that controls the growth and survival of prostate cells (19, 20). It has been suggested that androgen and PI3K/Akt pathways can compensate for each other in growth regulation and prostate development (21). The PI3K/Akt pathway may up-regulate AR activity by directly phosphorylating AR or through ß-catenin, an AR coactivator (22). Additionally, the small G protein, RhoA, which belongs to the Rho family of guanosine triphosphatases (GTPases), is an important intracellular signaling protein that controls diverse cellular functions related to prostate gland development. One of the possible mechanisms may involve Akt, because inhibition of RhoA activation results in the activation of Akt (23). Thus, understanding the regulation of these signaling pathways by E6-AP may be crucial to elucidating the role of E6-AP in prostate development.

The role of E6-AP in the AR transactivation pathway and prostate gland development has not been fully explored. Evidence from E6-AP-null mice suggests that E6-AP is involved in the various aspects of reproduction. E6-AP-null mice show reduced fertility that is attributed to defects in sperm production and ovulation. Furthermore, the induction of prostate gland growth by testosterone administration in E6-AP-null mice was significantly reduced (24). These observations suggest that ablation of E6-AP expression affects the physiological parameters associated with male and female sex steroid hormone action. The poorly responding prostate gland after hormone restimulation in E6-AP-null mice strongly supports the idea that E6-AP is involved in the AR transactivation pathway and in the proper development and growth of the prostate gland. To understand the mechanisms by which E6-AP exerts its effects on prostate gland development and growth, we carried out in vitro and in vivo studies. We found that the total prostate wet weights of E6-AP-null mice were less than those in wild-type animals, suggesting that E6-AP-null mice have smaller prostate glands than wild-type normal mice. We also report that E6-AP interacts with AR in the presence of hormone to enhance the transactivation functions of AR. To examine the in vivo role of E6-AP in prostate gland growth and development, we examined the expression patterns of E6-AP, AR, probasin, and several related proteins in prostate glands of E6-AP-null (–/–), E6-AP heterozygous (+/–), and wild-type (+/+) mice. Because E6-AP was reported to promote the degradation of p53 via the ubiquitin-proteasome pathway (25, 26), we analyzed the expression profile of p53 and p53-regulated genes in prostate glands of E6-AP-null mice. Moreover, another nuclear hormone receptor coactivator, SRC-3, can modulate Akt levels and activity in a prostate cancer cell line (27), which is critical for the survival of prostate cells. Hence, we also examined the possible role of E6-AP in the regulation of Akt signaling and found that loss of E6-AP decreased Akt levels, probably due to increased activity of RhoA, a putative negative regulator of Akt, which inhibits AR transactivation and promotes prostate cell apoptosis. Overall, our data reveal a key physiological role of E6-AP in the development of the prostate gland.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
E6-AP Is Required for the Development of Normal Prostate Gland
We have previously reported that the loss of E6-AP significantly reduced the induction of prostate gland growth in castrated mice by testosterone (24). It is of interest to determine whether E6-AP is involved in the growth of the prostate gland in normal unchallenged mice. The total prostate gland was dissected, and the wet weights of prostate glands from wild-type and E6-AP knockout mice were determined. As shown in Fig. 1AGo, the wet weight of the prostate gland from E6-AP knockout mice was approximately 40% less compared with that from the wild-type controls. These data indicate that E6-AP is involved in the development of normal prostate gland and growth. Because the loss of E6-AP impairs prostate gland development, and both stromal and epithelial cells play a major role in the normal development of the prostate gland, we next examined the expression pattern of E6-AP in prostate gland. As shown in Fig. 1BGo, our data demonstrate that E6-AP is predominantly expressed in epithelial cells. These observations suggest that the loss of E6-AP might influence prostate gland development by affecting the epithelial cells of the prostate.


Figure 1
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  		Fig. 1. Prostate Gland Development Is Impaired in E6-AP-Null Mice

A, The wet weights of total prostate gland corrected to the body weight were calculated for 12-wk-old wild-type (+/+; n = 10) and E6-AP knockout (–/–; n = 15) male mice, and data were plotted as 100% for wild-type mice. The wet weights of E6-AP-null prostate gland were significantly reduced (~40%) compared with those of the wild-type animals. B, Immunohistochemical analysis of E6-AP in mouse prostate tissue. E6-AP is predominantly expressed in epithelial compartment of mouse prostate. Paraffin-embedded total prostate tissue from wild-type mice was processed for immunohistochemistry using anti-E6-AP antibody. Positive nuclei are stained brown, and nonexpressing nuclei are purple.

 
E6-AP Interacts with AR in a Ligand-Dependent Manner
To determine whether E6-AP interacts with AR to modulate prostate gland development, we examined the ability of E6-AP to interact with AR in a hormone-dependent manner in glutathione-S-transferase (GST) pulldown assays. [35S]Methionine-labeled AR protein was incubated with either control protein (GST only) or GST-E6-AP protein in both the absence and presence of hormone (R1881). Figure 2AGo depicts a very weak interaction of E6-AP with AR in the absence of hormone. However, a significant level of E6-AP interaction with full-length AR was observed in the presence of hormone (R1881). Under similar conditions, AR did not interact with control GST protein. These findings suggest that E6-AP and AR directly interact, and this interaction is enhanced in the presence of hormone.


Figure 2
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  		Fig. 2. E6-AP Acts as a Coactivator of AR

A, In vitro interaction of E6-AP with AR. The human AR protein was synthesized in vitro using the TNT-coupled reticulocyte lysate system. AR protein was then incubated with its synthetic ligand (R1881) at 10–7 M or vehicle (ethanol) and incubated with GST-E6-AP fusion protein that was bound to glutathione-Sepharose beads. The glutathione-bound proteins were separated on SDS-PAGE, followed by autoradiography. Twenty percent of the TNT reaction was used as input, and GST alone was used as a negative control. B, E6-AP modulates the transcriptional activity of endogenous AR in LNCaP cells. LNCaP cells were transiently transfected with the AR response element (ARE) linked to the luciferase reporter gene and E6-AP expression vector. Four hours after transfection, cells were treated with either hormone R1881 (10–9 M) or ethanol vehicle. After 24 h, cells were harvested and assayed for luciferase activity. Data are presented as fold activation relative to the activity observed with hormone-treated cells containing endogenous levels of E6-AP. C, E6-AP modulates the transcriptional activity of transiently expressed AR in PC3 cells. PC3 cells were transfected with ARE-luciferase reporter, E6-AP expression vector, and AR expression vector. Then cells were treated with hormone R1881 (10–9 M) or ethanol vehicle as described above. Twenty-four hours later, cells were harvested and assayed for luciferase activity. Data are presented as fold activation relative to the activity of AR observed with hormone-treated cells containing endogenous levels of E6-AP.

 
E6-AP Modulates the Transcriptional Activity of AR
Having established an in vitro interaction between AR and E6-AP, the possibility that E6-AP is involved in AR-dependent activation of target gene expression was explored by transient transfection assays in an AR-positive prostate cancer cell line, LNCaP. The LNCaP cells were cotransfected with a luciferase reporter plasmid containing androgen response element, with or without an expression vector for E6-AP. Figure 2BGo suggests that E6-AP had no significant effect on the transactivation function of AR in the absence of hormone; however, E6-AP significantly (~4- to 6-fold) enhanced the hormone-dependent transcriptional activity of AR, indicating a strong role of E6-AP in AR transactivation. To confirm that E6-AP is involved in AR-dependent gene transcription, we also examined the ability of E6-AP to enhance AR function in an AR-negative prostate cancer cell line, PC3. Like LNCaP cells, E6-AP was able to stimulate the hormone-dependent transcriptional activity of exogenously expressed AR in PC3 cells (Fig. 2CGo). Similarly, E6-AP was able to enhance AR transactivation function in HeLa cells (data not shown). Together, our data demonstrate that E6-AP physically interacts with AR and enhances the transcriptional activity of AR approximately 3- to 6-fold in a hormone-dependent manner.

Reduction of Endogenous E6-AP Levels Reduces Transcriptional Activity of AR
To confirm that E6-AP is indeed required for AR transactivation, we knocked down the endogenous expression of E6-AP in LNCaP cells by small interfering RNA (siRNA). LNCaP cells were transiently transfected with siRNA directed against E6-AP along with AR-responsive reporter plasmid. Introduction of E6-AP siRNA in LNCaP cells resulted in significantly reduced levels of E6-AP expression compared with control cells that were transfected with nonspecific siRNA [glyceraldehyde-3-phosphate dehydrogenase (GAPDH); Fig. 3AGo]. We also observed that depletion of endogenous E6-AP resulted in a reduction of hormone-dependent transactivation of AR by an average of 84%, as measured by luciferase activity (Fig. 3BGo). E6-AP-specific siRNA also reduced the basal activity of AR. This basal activity of AR may be due to incomplete stripping of androgens from serum. These data confirm that E6-AP is required for the proper transactivation of AR.


Figure 3
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  		Fig. 3. Depletion of Endogenous E6-AP Affects the Transcriptional Activity of AR

A, LNCaP cells were transfected with ARE-luciferase reporter plasmid in the presence of either siRNA directed against GAPDH (negative control) or siRNA directed against E6-AP. Then cells were treated with hormone-R1881 (10–7 M) or ethanol vehicle. Twenty-four hours later, cells were harvested and analyzed for E6-AP expression by Western blot using anti-E6-AP-specific antibodies. ß-Actin antibodies were used to confirm equal loading. B, Luciferase activity was measured in cell lysate in the presence of GAPDH siRNA and E6-AP-specific siRNA. Data are presented as fold activation relative to the activity of AR observed with hormone-treated cells transfected with GAPDH siRNA.

 
E6-AP Interacts with and Enhances the Transcriptional Activity of the Amino Terminus of AR
Because E6-AP interacts with full-length AR in a hormone-dependent manner, it was of interest to determine whether the amino terminus ({Delta}C) or carboxyl terminus ({Delta}N) of AR is involved in E6-AP-mediated coactivation. The [35S]methionine-labeled {Delta}C or {Delta}N region of AR protein was incubated with control protein (GST) and GST-E6-AP protein. Figure 4AGo depicts a significant level of E6-AP interaction with the amino-terminal region (AR-{Delta}C) of the AR. In contrast, no significant interaction was observed with the carboxyl-terminal region (AR-{Delta}N) of the AR in the presence or absence of hormone. Our findings indicate that E6-AP and AR directly interact with each other via the amino-terminus of the AR. Because E6-AP modulates the transcriptional activity of full-length AR in a hormone-dependent manner, we determined whether E6-AP retained the transactivation function during its interaction with the amino-terminus region of the truncated AR receptor. HeLa cells were cotransfected with expression plasmids for the AR-{Delta}C region and the AR-{Delta}N region of the AR along with androgen-responsive luciferase reporter gene, with or without E6-AP expression vector. In the absence of the amino-terminus region ({Delta}N), the AR loses all of its transcriptional activity, and addition of E6-AP has no significant effect on the expression of the reporter gene (Fig. 4BGo). However, in the absence of the carboxyl terminus, the truncated AR shows a constitutive transcriptional activity, and it has no significant hormone dependency. E6-AP coexpression further increased the transcriptional activity of the amino-terminus region ({Delta}C) of the AR by 9-fold compared with activity in the absence of E6-AP (Fig. 4BGo). To confirm that the amino-terminal region of AR is the target of the E6-AP coactivation function, we replicated the experiment in a prostate cancer cell line, PC3. Like HeLa cells, E6-AP had minimal coactivation effect on the carboxyl-terminal region of the AR, whereas it stimulated the transcriptional activity of the amino-terminal region of the AR in PC3 cells several fold (data not shown). These results suggest that the amino-terminal region of the AR is the major target of E6-AP for interaction and coactivation function.


Figure 4
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  		Fig. 4. E6-AP Interacts with the Amino Terminus of AR to Modulate the Transcriptional Activity of the Receptor

A, In vitro interaction of E6-AP with the amino terminus of AR (AR-{Delta}C) and the carboxyl terminus of AR (AR-{Delta}N) was carried in GST pulldown assays. 35S-Labeled AR-{Delta}C and AR-{Delta}N proteins were synthesized using the TNT-coupled kit. The labeled proteins were then incubated with GST-E6-AP fusion protein that was bound to glutathione-Sepharose beads. The glutathione-bound proteins were analyzed on SDS-PAGE, followed by autoradiography. Twenty percent of the TNT reaction was used as input, and GST alone was used as a negative control. B, HeLa cells were cotransfected with AR response element fused with luciferase reporter gene along with the expression plasmids for amino-terminal deletion mutant (AR-{Delta}N) or carboxyl-terminal deletion mutant (AR-{Delta}C) of AR and E6-AP. After transfection, cells were treated with either R1881 (10–9 M) or ethanol and incubated for an additional 12–16 h. Luciferase activity was assayed, and data are presented as fold activation relative to the activity of AR observed with hormone-treated cells containing endogenous levels of E6-AP.

 
In VivoRecruitment of E6-AP onto the AR-Responsive Promoter
To better understand the coactivation function of E6-AP, we employed a chromatin immunoprecipitation (ChIP) assay to examine the recruitment of E6-AP to AR-responsive prostate-specific antigen (PSA) promoter. Formaldehyde-cross-linked chromatin complexes were immunoprecipitated with antibodies for AR and E6-AP from LNCaP cells in the presence or absence of R1881. Our ChIP analyses demonstrated the hormone-dependent recruitment of AR and E6-AP to AR-responsive PSA promoter in vivo (Fig. 5Go).


Figure 5
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  		Fig. 5. E6-AP Is Recruited onto AR-Responsive Promoter

ChIP was performed using LNCaP cells in the presence or absence of R1881 (10–7 M). Primers specific for the PSA promoter were used to amplify the genomic DNA associated with AR and E6-AP in LNCaP cells. In these experiments, 0.1% input DNA was used in the control lanes (Input).

 
E6-AP Also Coactivates AR when AR Is Activated by the Intermediate Compounds of the Testosterone Biosynthetic Pathway
To determine the effect of E6-AP on AR transactivation in the presence of androgen biosynthetic intermediates, we performed transient cotransfection experiments in HeLa cells. We examined the coactivation activity of E6-AP on AR in response to stimulation by ligands such as androstene-3ß,17ß-diol (Adiol), dehydroepiandrosterone (DHEA), and androstenedione as well as the AR antagonist casodex. We found that E6-AP dose-dependently coactivated AR activity in response to stimulation by androstenedione (Fig. 6AGo) and Adiol (Fig. 6BGo), but not DHEA (Fig. 6CGo). Furthermore, the AR antagonist casodex competitively inhibited the effect of androstenedione on AR transactivation coactivated by E6-AP in a dose-dependent manner (Fig. 6DGo). Transactivation of AR by androstenedione and Adiol cannot be attributed to the conversion of these hormone intermediates to testosterone and dihydrotestosterone, because HeLa cells lack the enzymes of androgen biosynthesis. Our data reveal that E6-AP enhances the transactivation function of AR not only in the presence of androgens, but also in the presence of androgen biosynthetic intermediates such as androstenedione and Adiol.


Figure 6
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  		Fig. 6. E6-AP Also Coactivates AR when AR Is Activated by Intermediate Metabolites of Testosterone Biosynthetic Pathway

HeLa cells were cotransfected with the AR-responsive reporter MMTV-Luc, AR expression vector, and empty control vector or E6-AP expression vector. Then the cells were treated with androstenedione (A), Adiol (B), or DHEA (C) for 24 h, followed by luciferase assay. Data are plotted as relative light units (RLU) per microgram of protein. D, E6-AP fails to coactivate AR function when it is bound to its antagonist, casodex. HeLa cells were transfected as described above and treated with androstenedione and various concentrations of casodex for 24 h, followed by luciferase assay. Data are plotted as RLU per microgram of protein.

 
E6-AP Modulates the Protein Levels and Function of AR
Our results suggest that E6-AP interacts with AR and modulates AR-dependent gene transcription. Because E6-AP is an E3 ubiquitin-protein ligase enzyme (13), and AR is a target of the ubiquitin-proteasome pathway (28), we examined the expression levels of AR in the prostate glands of wild-type (+/+), E6-AP heterozygous (+/–), and E6-AP-null (–/–) mice using Western blot analysis. As expected, no E6-AP expression was detected in E6-AP homozygous null prostate glands, whereas lower expression of E6-AP was observed in E6-AP heterozygous prostate glands compared with that in wild-type prostate glands (Fig. 7AGo). Furthermore, the levels of AR protein were elevated in E6-AP-null prostate glands compared with wild-type and E6-AP heterozygous prostate glands (Fig. 7AGo), implying that E6-AP modulates the protein levels of AR in the prostate gland. The higher levels of AR protein in E6-AP-null prostate gland led us to investigate the levels of the AR target protein, probasin (29). Our results indicate that the level of probasin, which is a direct target of AR, is reduced in E6-AP-null prostate gland despite higher levels of AR, suggesting that E6-AP is required for the proper transactivation function of AR (Fig. 7AGo). Alternatively, the elevated AR protein in E6-AP-null mice is transcriptionally inactive. To confirm that E6-AP modifies AR protein levels and its function, we generated E6-AP-overexpressing, stably transfected LNCaP cell lines using the Tet-off system. Figure 7BGo shows that the E6-AP-stable cell line has higher expression of E6-AP under inducible conditions [removal of doxycyclin (Dox) from medium] than wild-type LNCaP cells. As expected, AR levels were lower, and the levels of AR target gene PSA were higher in these stable cell lines (Fig. 7CGo), confirming that E6-AP modulates the protein levels as well as the transactivation functions of the AR.


Figure 7
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  		Fig. 7. Levels of AR and Its Target Genes Are Altered in E6-AP-Null Prostate Glands and Prostate Cancer Cell Lines that Stably Overexpress E6-AP

A, Total proteins from whole prostate extracts were resolved on SDS-PAGE and probed with anti-E6-AP, anti-AR, and antiprobasin antibodies. ß-Actin was used as a loading control. Multiple sets of siblings of male mice that were homozygous null (–/–), heterozygous-null (+/–), and homozygous wild type (+/+) were used for this study. B, LNCaP cells were generated that stably overexpress E6-AP under the Tet-off system. Two sets of plates of stably transfected LNCaP cells were treated with Dox (+Dox), which inhibits the expression of stably transfected E6-AP, or without Dox (–Dox), which induces the expression of stably transfected E6-AP. Whole-cell extracts were subjected to immunoblot analysis with antibodies against E6-AP. ß-Actin was used as a loading control. C, Stably transfected LNCaP cells in the absence of Dox (E6-AP overexpression) were treated with either vehicle (ethanol) or synthetic androgen (R1881). Whole-cell extracts were then analyzed for AR and PSA expression using anti-AR and anti-PSA antibodies. ß-Actin was used as a loading control. D, E6-AP promotes AR degradation through the ubiquitin-proteasome pathway. To study the in vitro ubiquitination and degradation of AR, 35S-labeled AR protein was synthesized in vitro with TNT-coupled rabbit reticulocyte extracts. The labeled AR protein was incubated with ATP, ubiquitin, UBA, and UBC in the absence or pres-ence of bacterially expressed E6-AP and proteasome proteasomeitor, MG132 (for 4 h). Reactions were terminated by adding sodium dodecyl sulfate-loading buffer, and results were analyzed by SDS-PAGE and autoradiography.

 
E6-AP Promotes AR Degradation
It is well known that AR is a target of the ubiquitin-proteasome pathway. Because our data suggest that the levels of AR protein are high in E6-AP-null prostate glands and low in stable cells that overexpress E6-AP; we propose that E6-AP promotes the degradation of AR via the ubiquitin-proteosome pathway. To test this hypothesis, we performed in vitro protein degradation and ubiquitination assay on AR protein in the absence and presence of E6-AP. As shown in Fig. 7DGo, E6-AP was able to promote AR degradation via the ubiquitin-proteasome pathway, and the proteasome inhibitor MG132 can block this degradation. These data confirm that E6-AP modulates protein levels of AR by promoting its degradation through the ubiquitin-proteasome pathway.

Levels of p53, p21, Bax, and Active Caspases Are Elevated in E6-AP-Null Prostate Gland
Because it has been shown that E6-AP promotes the degradation of p53, a tumor suppressor, via the ubiquitin-proteasome pathway and the levels of p53 are elevated in E6-AP-null brain (24), we examined the levels of p53 and its target proteins in E6-AP-null prostate. As shown in Fig. 8Go, the E6-AP-null and E6-AP heterozygous prostate glands exhibited higher expression of p53 compared with those in their normal littermates. These data confirm that p53 is also a target of E6-AP in the prostate gland. Because p53 is a key modulator of p21 expression, we examined the expression of p21 in E6-AP-null prostate. Figure 8Go demonstrates that the level of p21 protein was also increased in heterozygous and homozygous E6-AP-null prostate glands. These data suggest that the transactivation function of p53, by which it regulates the p21 expression, does not require E6-AP.


Figure 8
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  		Fig. 8. Levels of p53, p21, Bax, and Caspase-3 and -9 Are Altered in E6-AP-Null Prostate Glands

Total protein from whole prostate extracts was resolved on SDS-PAGE and analyzed by Western blot using antibodies against p53, p21, Bax, caspase-3, and caspase-9. ß-Actin was used as a loading control.

 
It has been established that p53 activates transcription of the proapoptotic protein Bax (30). The increased levels of p53 and the fact that the prostate gland is smaller in E6-AP-null mice prompted us to examine the expression levels of Bax in E6-AP-null prostate glands. Figure 8Go demonstrates that the level of Bax protein, which is a target of p53, is increased in E6-AP-null prostate glands. Additionally, we examined the levels of other proapoptotic proteins, such as caspase-3 and -9. As shown in Fig. 8Go, the level of active caspase-9 was higher in E6-AP-null prostate glands. Similarly, the level of active caspase-3 was higher in E6-AP-null prostate glands. These data suggest that the loss of E6-AP promotes apoptosis of prostate cells. To confirm that the loss of E6-AP in the prostate gland induces apoptosis, we also determined the level of apoptosis in E6-AP-null prostate glands by terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling (TUNEL) assay. Because there was no significant change in the apoptosis of stromal cells compared with epithelial cells, we quantified our results based on apoptosis in epithelial cells. Our results indicate that epithelial apoptosis was increased in E6-AP-null prostate glands compared with that in wild-type prostate (Fig. 9Go, A and B). These data indicate that E6-AP can modulate the growth of the prostate gland by controlling apoptosis of prostate epithelial cells. Taking into account the complex interplay between stromal and epithelial cells in the normal development of the prostate gland, it is important to consider the expression pattern of E6-AP in the prostate. Our data suggest that apoptosis occurs predominantly in epithelial cells (Fig. 9AGo), and this correlates with our observation that E6-AP was predominantly expressed in epithelial cells (Fig. 1BGo). Because AR is also expressed in epithelial cells, our results demonstrate that both E6-AP and AR are expressed in the same cell types. This, coupled with facts that E6-AP interacts with and efficiently coactiavates AR, and loss of E6-AP impairs prostate gland growth, make it an important regulator of AR activity in prostate epithelial cells.


Figure 9
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  		Fig. 9. Elevated Levels of Apoptosis in E6-AP-Null Prostate Glands

A, TUNEL assays to observe apoptotic cells in sections of wild-type and E6-AP-null prostate glands. Apoptotic cells were detected using the In Situ Cell Death Detection Kit (Roche) system according to the manufacturer’s instructions. The arrowheads indicate apoptotic cells. B, Prostate tissue sections from wild-type (+/+) and E6-AP homozygous knockout (–/–) mice were analyzed for apoptosis using the TUNEL assay. The number of TUNEL-positive epithelial cells in wild-type and E6-AP-null prostate glands was counted, and data are plotted as the percentage of TUNEL-positive cells. These data are representative of three sets of wild-type and null prostate glands. At least 1000 cells for each prostate gland were counted. C, Increased proliferation in E6-AP-overexpressing stable cell lines. LNCaP cells stably transfected with E6-AP were treated with (+) or without (–) Dox for 72 h, harvested, fixed with 70% ethanol, stained with propidium iodide, and processed for FACS analysis. The experiment was repeated three times with identical results. Data are plotted as the percentage of S phase cells.

 
Because the loss of E6-AP induces apoptosis in prostate tissues, we wanted to determine whether overexpression of E6-AP induces proliferation of prostate cancer cells. We performed propidium iodide staining to analyze the DNA content of E6-AP stably transfected cell lines. Cells treated with Dox did not express exogenous E6-AP and served as controls to untreated cells, which overexpress E6-AP (Fig. 7BGo). As shown in Fig. 9CGo, there was an increase in the number of cells in S phase in Dox-free cells compared with Dox-treated cells that did not overexpress E6-AP. These data suggest that overexpression of E6-AP leads to proliferation, which correlates with the fact that the loss of E6-AP leads to apoptosis.

Levels and Activity of the Components of PI3K/Akt Pathway Are Decreased in E6-AP-Null Mice
In addition to androgen signaling, which plays an essential role in the growth and development of the prostate gland, the PI3K/Akt pathway is involved in AR transactivation and prostate cell growth (19, 20, 31). To determine whether E6-AP also contributes to the growth and development of the prostate gland by affecting the components of the PI3K/Akt pathway, we examined the expression levels of total Akt and phosphorylated Akt in E6-AP-null prostate glands and control wild-type prostate glands. As shown in Fig. 10AGo, the levels of total Akt were decreased in E6-AP-null and heterozygous prostate glands compared with those in control wild-type prostate. Similarly, the levels of phosphorylated (activated) Akt at Ser473 were significantly lower in E6-AP-null prostate glands compared with control normal glands (Fig. 10AGo). We also found that the levels of phosphorylated Akt at Thr308 were significantly low in E6-AP-null prostate glands (data not shown). These data suggest that E6-AP is also involved in the modulation of Akt activity and protein levels.


Figure 10
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  		Fig. 10. Levels and Activity of PI3K/Akt Pathway Components and Total and Active RhoA Are Altered in E6-AP-Null Mice

A, Total protein from whole prostate glands of E6-AP-null (–/–), heterozygous (+/–), and wild-type (+/+) mice were isolated and resolved on SDS-PAGE and probed with anti-PI3K, anti-Akt/PKB, antiphosphorylated Akt (Ser473), and anti-RhoA antibodies. ß-Actin was used as a loading control. B, Total protein from whole prostate glands of E6-AP-null (–/–) and wild-type (+/+) mice was used to determine the levels of active RhoA. The extracts from wild-type (+/+) and E6-AP-null (–/–) prostate glands were incubated with GST-fused RhoA-binding domain (GST-RBD) or GST alone (GST) as a negative control. The amount of active RhoA was analyzed by Western blot using anti-RhoA antibody. The input lane represents the amount of total RhoA.

 
Because Akt is a downstream effector of PI3K enzyme, and activation of Akt (phosphorylation) is dependent on PI3K activation, we probed the levels of PI3K enzyme (regulatory subunit p85) in E6-AP-null prostate glands. Figure 10AGo demonstrates that similar to Akt, the levels of PI3K were reduced in E6-AP-null prostate glands compared with control wild-type glands. E6-AP heterozygous prostate glands exhibited an expression intermediate between wild-type control and E6-AP-null glands (Fig. 10AGo). These data demonstrate that lower levels of phosphorylated Akt in E6-AP-null prostate glands is at least in part due to lower protein levels of PI3K enzyme, which is perhaps regulated by E6-AP.

Recently, it has been demonstrated that activated RhoA can negatively modulate Akt signaling pathway via protein kinase C{zeta}. Inhibition of RhoA activation results in the activation of Akt, leading to enhanced cell survival of murine prostate cancer cells (23). Therefore, RhoA may also be directly involved in the development of prostate through the modulation of apoptosis. Because the levels of total Akt and phosphorylated Akt (activated) are reduced in E6-AP-null prostate glands, we considered it essential to analyze the RhoA protein level in E6-AP-null prostate glands, because RhoA can negatively modulate Akt signaling (23). Figure 10AGo demonstrates that the levels of total RhoA were significantly increased in E6-AP-null prostate glands compared with those in control normal and heterozygous animals. Our data also demonstrate that the levels of active RhoA were increased in E6-AP-null prostate compared with wild-type prostate (Fig. 10BGo). Collectively, these data suggest that E6-AP may also modulate the Akt signaling pathway by modulating the protein levels of active RhoA in E6-AP-null prostate glands.

To confirm the role of RhoA in the regulation of Akt activation in prostate cells, we treated LNCaP cells with C3 transferase, a specific inhibitor of RhoA function, and then examined the effect of inhibition of RhoA on the level of active Akt in LNCaP cells. Our data suggest that C3 transferase only marginally increased total Akt expression, but strongly increased the phosphorylated form of Akt (active). In contrast, the levels of PI3K and RhoA proteins remained unchanged throughout C3 transferase treatments (Fig. 11AGo). Thus, inactivation of RhoA by C3 transferase increased the levels of active Akt, suggesting a negative modulation of the Akt pathway by endogenous RhoA in LNCaP cells.


Figure 11
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  		Fig. 11. RhoA Is a Negative Regulator of Akt and AR Activity

A, Inhibition of RhoA with C3 transferase significantly increases the level of activated Akt (phosphorylated). LNCaP cells were cultured for 24 h and exposed to either 5 µg/ml C3 transferase or vehicle control for 24 h. After C3 transferase treatment, proteins were analyzed by Western blot using anti-RhoA, anti-total Akt, antiphosphorylated Akt, and anti-PI3K antibodies. ß-Actin was used as a loading control. B, RhoA inhibits AR transactivation. LNCaP cells (passages 31–35) were transfected with the reporter plasmid MMTV-luc control plasmid, or plasmid encoding the constitutively active form (RhoA G14V) or the dominant-negative form (RhoA T19N) of RhoA GTPase. Cells were treated with ethanol (vehicle) or 5 nM of the synthetic androgen R1881. The luciferase activities of cell lysates were measured, and data are plotted as relative luciferase activity. The results shown represent an experiment performed in triplicate and repeated twice with similar results.

 
RhoA Is a Negative Regulator of AR Function
Because Akt can transactivate the AR in LNCaP cells, we were interested to determine whether inhibition of Akt activity by RhoA can modulate AR function. LNCaP cells were transfected with AR reporter plasmid along with the constitutively active form (RhoA G14V) or the dominant-negative form (RhoA T19N) of RhoA GTPase (Fig. 11BGo). Constitutively active RhoA was able to reduce the transactivation functions of AR, whereas the dominant-negative form of RhoA enhanced the androgen-dependent transactivation functions of AR (Fig. 11BGo). These results suggest that RhoA can also negatively regulate prostate cell growth by exerting its effect on AR signaling.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Our previous observations of E6-AP-null mice suggested that androgen-stimulated prostate gland growth in castrated animals is impaired (24). The data presented in this study from noncastrated E6-AP-null mice indicate that E6-AP plays a vital role in prostate growth and development. In this report we demonstrate that E6-AP interacts with AR in vitro in a hormone-dependent manner, and it coactivates the hormone-dependent transcriptional activity of AR. These data are consistent with our previously published data indicating that E6-AP only interacts with receptors in the presence of hormones and modulates their transactivation function (10). Our data also demonstrate that E6-AP, like the other AR coactivators, interacts with AR via the amino terminus, and this interaction is responsible for E6-AP modulating the transcriptional activity of AR. Our ChIP analysis demonstrates the recruitment of E6-AP onto AR-responsive promoters, indicating that E6-AP physically associates with the PSA promoter. These results are consistent with previously published findings, which suggest that coactivator proteins are recruited by receptors to target promoters in a hormone-dependent manner and regulate target gene transcription.

It has been postulated that coactivators can change the AR response to hormones other than androgen (32). In this study we have demonstrated that E6-AP can coactivate AR in the presence of different androgen biosynthetic intermediates. In the presence of E6-AP, the unfavored hormones, androstenedione and Adiol, enhanced the transactivation functions of AR. Given the androgen-dependent nature of prostate epithelial cells, these findings indicate that overexpression of E6-AP may aberrantly enhance AR activity and may result in increased cell proliferation in prostate epithelial cells. Our data also suggest that in hormone refractory cancers, E6-AP keeps AR transcriptionally active even in the absence of androgens, because E6-AP can coactivate AR in response to the stimulation by androstenedione and adiol. Because casodex dose-dependently inhibits AR transactivation in the presence of androstenedione, this suggests that androstenedione exerts its effect via AR.

The smaller size of the prostate gland in E6-AP-null mice could potentially be due to reduced cell growth, decreased cell proliferation, or increased apoptosis in the developing gland. In normal prostate, the processes of cell proliferation and differentiation are regulated to a large extent by androgens and AR (1, 15). Our data demonstrate that E6-AP regulates protein levels and the function of AR in prostate cells. It has been previously shown that in prostate cancer cell lines AR is degraded by the ubiquitin-proteasome pathway (28, 33, 34). In this report we have demonstrated for the first time that AR is degraded in vivo in mouse prostate gland by E6-AP, an E3 ubiquitin ligase. These data are consistent with our previously published report that down-regulation of E6-AP in prostate carcinoma is associated with up-regulation of AR protein levels (35). Previous reports have shown that the ubiquitin-proteasome-dependent degradation of steroid hormone receptors, including AR, is required for their proper functioning (12, 33, 34). It has been suggested that ligand binding is one of the signals for steroid hormone receptor degradation (36). However, unlike other receptors, AR is stabilized upon binding to its ligand. A few papers have been published about the stability of AR in animal models (such as mice and rats), but the data are controversial (37, 38, 39). Studies performed in prostate cancer cell lines have shown that proteasome activity is essential for AR transactivation (33, 34). Our data from a prostate cancer cell line and an in vivo mouse model complement these reports. Although androgen binding to AR itself may not be a signal for its degradation, it is still possible that transcriptionally active receptor that is associated with E6-AP and other cofactors may be target of degradation via the ubiquitin-proteasome pathway along with the AR transcription complex, and this degradation is necessary for the transactivation functions of AR. Our in vivo and in vitro data are consistent with and supportive of this hypothesis, in view of the fact that the levels of AR target gene probasin are decreased in E6-AP-null prostate glands. Furthermore, AR levels are lower, and probasin levels are higher, when levels of E6-AP are increased. Thus, one of the mechanisms by which E6-AP controls prostate gland growth is by regulation of AR protein levels and transactivation functions.

It has been suggested that besides androgen signaling, the PI3K/Akt signaling pathway also plays a role in the development of the prostate gland (19, 20). The PI3K/Akt signaling pathway is involved in AR transactivation in an androgen-independent manner. Deciphering the link between these signaling pathways is critical for elucidating the role of E6-AP in prostate gland development. Because SRC-3 can modulate the protein levels of the components of the PI3K/Akt pathway (27), we examined the possible role of E6-AP in regulation of the PI3K/Akt pathway. Our data demonstrate that the levels of PI3K, total Akt, and phosphorylated Akt are decreased in E6-AP-null prostate glands, suggesting that, like SRC-3, E6-AP modulates the protein levels of the components of the PI3K/Akt pathway. However, we do not know whether E6-AP regulates the PI3K/Akt pathway directly or indirectly. Because E6-AP acts as a coactivator, it is possible that E6-AP may be controlling the expression of PI3K/Akt at the transcription level.

Another factor that may be involved in the regulation of Akt signaling by E6-AP is the small G protein, RhoA. Several studies have shown that RhoA activation is necessary for the growth of normal and cancer cells (40, 41). It has also been shown that RhoA can promote prostate cancer cell apoptosis by inhibiting Akt signaling pathway via the protein kinase C{zeta} (23). Our data demonstrate that the levels of total RhoA and active RhoA are increased in E6-AP-null prostate gland. It is possible that RhoA may affect the Akt pathway by inducing the inactivation of Akt in the absence of E6-AP, which subsequently alters prostate gland development. This possibility was supported by the fact that the inhibition of RhoA activity increased the levels of phosphorylated (active) Akt. LNCaP cells are phosphatase and tensin homolog negative, and Akt is constitutively active in these cells (42); therefore, it is possible that RhoA regulation of Akt in these cells is independent of phosphatase and tensin homolog. Because the levels of active RhoA are increased in E6-AP-null prostate glands, we propose that increased levels of RhoA negatively regulate Akt function and exert its effect on prostate gland development. Moreover, our RhoA activation assay demonstrated that the increased RhoA levels result in increased RhoA activation. However, we do not know whether E6-AP regulates RhoA levels directly or indirectly. E6-AP may control the expression of RhoA by modulating its degradation via the ubiquitin-proteasome pathway. In this study we also show that RhoA acts as a negative regulator of AR signaling; therefore, it is possible that lower AR activity in E6-AP-null mice may also be due to higher RhoA levels.

Initially, it was suggested that E6-AP functions as an E3 ubiquitin-protein ligase for p53 degradation via the proteasome pathway, only in association with the E6 protein of papillomavirus (13). Evidence concerning whether E6-AP targets p53 for degradation in the absence of E6 protein has been conflicting. However, elevated levels of p53 expression are detected in the brain of E6-AP-null mice, suggesting that E6-AP contributes to the regulation of p53 in the brain (24). In this study we also demonstrate that levels of p53 are elevated in E6-AP-null prostate glands, suggesting that E6-AP contributes to the regulation of p53 protein levels in prostate gland. Furthermore, our data demonstrate that, unlike AR, E6-AP is not required for the transcriptional activity of p53, because the levels of the p53 downstream target proteins p21 and Bax are also elevated in E6-AP-null prostate gland. It has been shown that p21 is also a target of AR (43), because AR function is decreased in E6-AP-null prostate; it is unlikely that AR has a major effect on p21 expression. The elevated levels of p21 in E6-AP-null prostate are primarily due to the increased levels of p53.

The smaller size of the prostate gland and higher levels of p53 protein in E6-AP-null mice support the idea that the p53 protein may control prostate gland growth by modulating the expression levels of proapoptotic proteins and apoptosis. This possibility is supported by the fact that protein levels of Bax and active caspases are elevated in E6-AP-null prostate glands. Moreover, epithelial apoptosis was increased in E6-AP-null prostate glands. This suggests that one of the possible mechanisms by which E6-AP controls prostate gland growth and development is by modulating p53-induced apoptosis.

In conclusion, the results presented in this study demonstrate that E6-AP controls the growth of the prostate gland by modulating several major cellular pathways, such as AR, PI3K/Akt, and RhoA signaling pathways and the p53-mediated apoptotic pathway.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Animals
All animals were maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. E6-AP-null mice were developed as described previously (24) and were maintained in a C57BL/6 background. The progeny of mating between two heterozygous parents were screened for the desired genotype using PCR as described previously (24). Twelve- to 14-wk-old male E6-AP-null mice were used to isolate whole prostate glands for wet weight analysis and protein immunoblot analysis. Mice were first killed by CO2 asphyxiation, and the whole genitourinary tract was isolated. All lobes of the prostate were harvested by microdissection under a dissecting microscope, immediately frozen in liquid nitrogen, and pulverized to isolate protein or stored at –80 C. C57BL/6 mice used for breeding to maintain the line were purchased from Charles River Laboratories, Inc. (Wilmington, MA).

Cell Culture
HeLa and PC3 cells were maintained in DMEM containing 10% fetal bovine serum. LNCaP cells and E6-AP-stable cell lines were maintained in RPMI 1640 medium with 10% fetal bovine serum. All transfections were carried out with cells grown in medium supplemented with 10% dextran-coated charcoal-stripped serum.

Plasmid Construction
The androgen-responsive reporter pARE-TATA-Luc (ARE, androgen response element; Luc, luciferase; p, plasmid) and pMMTV-Luc (MMTV, mouse mammary tumor virus) have been previously described (44, 45). The mammalian expression plasmids for full-length androgen receptor (pCR3.1.AR), the amino-terminal-truncated androgen receptor (pCR3.1.AR-{Delta}N), and the carboxyl-terminal-truncated androgen receptor (pCR3.1.AR-{Delta}C) have been described previously (46, 47). E6-AP expression (pBK-RSV-E6-AP) and GST fusion plasmids have also been previously reported (10). The constitutively active form (RhoA G14V) and the dominant-negative form (RhoA T19N) of RhoA GTPase were obtained from UMR cDNA Resource Center (www.cdna.org). The pRevTRE-E6-AP vector for developing stable cell lines was generated as follows. The BamHI-HindIII fragment of E6-AP isolated from pGEM-E6-AP was inserted into the corresponding sites of the pRevTRE vector.

Transfections
Cells were plated in six-well plates 24 h before transfection at a density of 3 x 105 cells/well in the appropriate medium with 10% dextran-coated charcoal-stripped serum. Cells were transfected with FuGene 6 transfection reagent (Roche, Indianapolis, IN). Four hours after transfection, cells were treated with the relevant hormones and were harvested 16–24 h later to perform the luciferase assay. To generate siRNA against E6-AP, the siRNA target finder program from Ambion, Inc. (Austin, TX), was used. GAPDH (control) siRNA was purchased from Ambion, Inc. The oligonucleotides used were as follows: for E6-AP siRNA, 5'-AATGAGTTTTGTGCTTCCTG-3'; and for GAPDH siRNA, 5'-GGATATTGTTGCCATCA TT-3'.

Luciferase Assay
Transfected cells were harvested in luciferase lysis buffer (Promega Corp., Madison, WI), and 100 µl of each lysate was analyzed for luciferase activity using the Promega luciferase assay system according to the manufacturer’s instructions. Protein concentrations of lysates were estimated using the Bradford protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA).

Protein Extraction from Prostate Tissue and Cultured Cells
The frozen whole prostate specimens from wild-type (+/+), E6-AP heterozygous (+/–), and E6-AP null (–/–) mice were pulverized in liquid nitrogen using a pestle and mortar, and the tissue powder was immediately processed for protein extraction as follows: 2–3 vol/wt RIPA lysis buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.5% sodium deoxycholate, 1% IGEPAL CA-630, and 0.1% sodium dodecyl sulfate] containing phenylmethylsulfonylfluoride (1 mM) and 1x protease inhibitor mixture (Sigma-Aldrich Corp., St. Louis, MO) was added to the frozen tissue powder in a 2-ml microfuge tube. The tissue powder in RIPA buffer was homogenized on ice using 5- to 10-sec pulses of a PowerGen 125 homogenizer (Fisher Scientific, Pittsburgh, PA) with a 5-mm diameter generator probe, to disintegrate any larger tissue pieces. The tissue homogenates were placed on ice for 20 min, then cleared by centrifugation at 12,000 x g for 10 min at 4 C. The supernatants were collected and frozen at –80 C until used for analysis. The protein concentrations of lysates were measured with the Bio-Rad protein assay kit using a 1:20 to 1:50 dilution of lysate.

Protein extraction from cultured cells was carried out as follows. Cells were grown for 24 h, then washed in TEN buffer [40 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 150 mM NaCl] and lysed in ice-cold RIPA buffer by pipetting up and down. Thereafter, cell lysates were placed on ice for 30 min, then cleared by centrifugation at 12,000 x g for 10 min at 4 C. The supernatants were collected and frozen at –80 C until used for analysis. The protein concentrations of lysates were measured using the Bio-Rad protein assay kit.

Western Blot Analysis
Twenty-five to 50 µg total protein from each sample was resolved on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred onto nitrocellulose membranes (Protran, Schleicher & Schuell, Inc., Keene, NH). Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline [20 mM Tris base (pH 7.5) and 150 mM NaCl] containing 0.05% Tween 20 (TBS-T), then probed with the primary antibody. The following primary antibodies were diluted in 1% nonfat dry milk in TBS-T as indicated and used for immunoblotting: anti-p53 (FL-393; 1:500), anti-AR (C-19; 1:500), antiprobasin (I-17; 1:400), anti-PSA (C-19; 1:600), anti-p21 (C-19; 1:200), and anti-RhoA (26C4; 1:800; Santa Cruz Biotechnology, Inc., Santa Cruz, CA); anti-E6-AP (1:1000; gift from Dr. Norm J. Maitland, University of York, York, UK); anti-total Akt (1:1200), antiphospho-Akt (1:1000), anticaspase-3 (8G10; 1:1000), and anti-Bax (1:750; Cell Signaling Technology, Beverly, MA); anticaspase-9 (1:1000; Neo Markers, Fremont, CA); anti-PI3-Kinase and p85 (1:1000; Upstate Biotechnology, Inc., Lake Placid, NY); and anti-ß-actin (1:10,000; Sigma-Aldrich Corp.). After washing in TBS-T, membranes were incubated with their appropriate horseradish peroxidase-conjugated secondary antibodies (Bio-Rad Laboratories, Inc.) and developed using an enhanced chemiluminescence detection system (Amersham Biosciences, Arlington Heights, IL) according to the instructions of the manufacturer. Membranes were exposed to X-OMAT AR film (Eastman Kodak Co., Rochester, NY).

In Vitro Interaction Assay
Radiolabeled AR, AR-{Delta}C, and AR-{Delta}N proteins were synthesized using a rabbit reticulocyte-coupled in vitro transcription and translation (TNT) kit in the presence of [35S]methionine according to the manufacturer’s recommendations (Promega Corp.). GST-E6-AP and control GST proteins were expressed in Escherichia coli DH-5{alpha} cells and immobilized on glutathione-Sepharose beads. The glutathione-bound GST and GST-E6-AP were incubated with in vitro-synthesized AR, AR-{Delta}C, and AR-{Delta}N proteins in NETN buffer [100 mM NaCl, 1 mM EDTA, 20 mM Tris (pH 8.0), and 0.5% Nonidet P-40] for 2–3 h at room temperature or overnight at 4 C. After washing four times with NETN buffer, E6-AP-bound AR, AR-{Delta}C, and AR-{Delta}N proteins were extracted by boiling the Sepharose beads in 1x sodium dodecyl sulfate gel loading buffer and were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel, followed by autoradiograpy.

To examine the levels of active RhoA, Rho assay reagent (Upstate Biotechnology, Inc.), and a glutathione-agarose bound GST-tagged fusion protein corresponding to residues 7–89 of mouse Rhotekin Rho binding domain was used. This specifically binds to and precipitates GTP-Rho, but not GDP-Rho, from cell lysates. Briefly, 1 ml of the cell lysate was incubated with Rho assay reagent for 45 min at 4 C. Beads were pelleted, washed, and boiled with sample buffer; SDS-PAGE was run; and beads were blotted onto a membrane. The membrane was probed with anti-RhoA antibody (Cell Signaling Technology).

ChIP
LNCaP cells were used in ChIP analyses following a modified procedure based on previously described protocols (9). The DNA was purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA) and eluted in 30 µl H2O. Each PCR mixture contained 4 µl immunoprecipitate or 1.5 µl input, a 0.2-µM concentration of each primer, 0.4 mM deoxynucleoside triphosphate mixture, 1x titanium Taq PCR buffer (BD Clontech, Palo Alto, CA), and 1x titanium Taq DNA polymerase (BD Clontech) in a total volume of 25 µl. The primers for the PSA promoter were as follows: forward, 5'-TCTGCCTTTGTCCCCTAGAT-3'; and reverse, 5'-AACCTTCATTCCCCA GGACT-3'. PCR was performed for 35 cycles with 30 sec of denaturing at 94 C, annealing for 1 min at 60 C, and extension at 72 C for 1 min, followed by one cycle for 2 min at 72 C.

Immunohistochemistry
Paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated through graded alcohol. Antigen was retrieved by boiling the slides in the antigen-unmasking solution (Vector Laboratories, Inc., Burlingame, CA) in a microwave oven for 10 min, and endogenous peroxidase activity was quenched by a 10-min incubation of the sections in 3% hydrogen peroxide at room temperature. The sections were incubated overnight with 10% normal goat serum in TBS at 4 C to block the nonspecific immunoreactivity. Antibodies against E6-AP (a rabbit polyclonal antibody, donated by Dr. Norman J. Maitland) at dilutions of 1:200 were added to the sections and incubated for 1 h at room temperature in a humidity chamber. For detection of the immunoreactivity, sections were then incubated with biotinylated antirabbit antibody (Vector Laboratories, Inc.), washed in TBS, incubated with streptavidin-conjugated peroxidase (Vectastain ABC kit, Vector Laboratories, Inc.), washed, and developed with 3,3'-diaminobenzidine (DAB substrate kit, Vector Laboratories, Inc.) according to the manufacturer’s recommendations. Finally, the sections were counterstained with hematoxylin and coverslipped for bright-field microscopy after dehydration in graded alcohol and clearance in xylene. Negative controls were included in each experiment using nonimmune serum in replace of primary antibody.

TUNEL Assay
Apoptotic cells in sections of prostate glands were detected using the In Situ Cell Death Detection Kit (Roche) according to the manufacturer’s instructions. Briefly, paraffin-embedded sections were deparaffinized, rehydrated, and fixed. The tissues were permeabilized with proteinase K; the DNA strand breaks were end labeled with fluorescein-labeled nucleotide with terminal deoxynucleotidyl transferase. Incorporated fluorescein was detected by antifluorescein antibody conjugated with horseradish peroxidase. Labeled cells were detected with chromagen substrate 3,3'-diaminobenzidine. Finally, the sections were counterstained with hematoxylin and mounted. A quantitative evaluation of apoptotic cells was performed, and the results were expressed as TUNEL-positive cells per 100 epithelial cells.

In VitroSynthesis of AR and Ubiquitination Assay
In vitro synthesis of radiolabeled AR was performed using TNT-coupled rabbit reticulocyte extracts in the presence of [35S]methionine according to the manufacturer’s recommended condition (Promega Corp.). 35S-Labeled AR was incubated in the presence of E1, E2, with or without E6-AP, and MG-132 in a mixture containing 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 4 mM ATP, 10 mM MgCl2, 0.2 mM dithiothreitol, and 4 µg ubiquitin for 3 h at 30 C. Reactions were terminated by boiling samples in the presence of sodium dodecyl sulfate-loading buffer [100 mM Tris-HCl (pH 8.0), 200 mM dithiothreitol, 4% sodium dodecyl sulfate, 20% glycerol, and 0.2% bromophenol blue]. The reaction mixtures were resolved by 10% SDS-PAGE, and radiolabeled bands were visualized by autoradiography.

Establishment of E6-AP-Stable Cell Line
The Tet-off system was used to develop stable cell lines (BD Clontech). LNCaP cells were grown to approximately 80% confluence in RPMI 1640 medium and transfected with pRevTet-off IN vector using the FuGene 6 transfection reagent. After 2 d the cells were treated with 200 µg/ml G418 and grown for 2 wk; the medium was changed every 4 d along with G418. Selected colonies were isolated and grown separately. These clones were screened using pTRE2hyg-Luc as a reporter vector. Selected clones were used to create a double-stable cell line.

To develop a double-stable cell line, the pRevTet-off IN-stable cell line was transfected with pRevTRE-E6-AP, and after 2 d the cells were treated with 150 µg/ml hygromycin and grown for 2 wk, changing the medium every 4 d along with hygromycin. Selected colonies were isolated and grown separately. The clones were tested for E6-AP gene expression in the presence or absence of Dox. All double-stable clones were maintained in the presence of 75 µg/ml hygromycin. For experiments, cells were untreated, which causes overexpression of E6-AP, or were treated with Dox (2 µg/ml), which shuts down exogenous E6-AP, and served as controls for untreated cells.

Cell Cycle Analysis
LNCaP cells stably transfected with E6-AP were treated with or without Dox. Approximately 3 x 105 cells were trypsinized and washed once in PBS. They were then fixed in cold 70% ethanol and stored at 4 C overnight. Before staining, ethanol was aspirated and treated with PBS containing ribonuclease (1µg/ml) and propidium iodide (50 µg/ml) for 30 min at 37 C. Cell cycle analysis was carried out using a FACScan (BD Biosciences, Mountain View, CA), and analysis was performed with CellQuest Pro software (BD Biosciences).


   ACKNOWLEDGMENTS
 
We thank Drs Fred Pereira, David Lonard, and Dr. Kerry Burnstein and Sarath Dhananjayan for critical reading of the manuscript. We also thank Dr. Arthur L. Beaudet for providing the E6-AP-null mice.


   FOOTNOTES
 
This work was supported by grants from the National Institutes of Health (DK-56833 and DK-060907; to Z.N.) and the National Center for Research Resource (P20-RR-018759) and the Creighton Health Foundation (to Y.T.).

Current address of O.Y.K.: Department of Pharmacology, Creighton University, 2500 California Plaza, Omaha, Nebraska 68178.

First Published Online October 27, 2005

1 O.Y.K. and G.F. contributed equally to this work. Back

Abbreviations: Adiol, Androstene-3ß,17ß-diol; AR, androgen receptor; ARE, androgen response element; ChIP, chromatin immunoprecipitation; DHEA, dehydroepiandrosterone; Dox, doxycyclin; E6-AP, E6-associated protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione-S-transferase; GTPase, guanosine triphosphatase; Luc, luciferase; MMTV, mouse mammary tumor virus; p, plasmid; PI3K, phosphatidylinositol 3-kinase; PSA, prostate-specific antigen; siRNA, small interfering RNA; SRC, steroid hormone receptor coactivator; TBS-T, Tris-buffered saline containing 0.05% Tween 20; TNT, transcription and translation; TUNEL, terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling; UBA, E1 ubiquitin-activating enzyme; UBC, E2 ubiquitin-conjugating enzyme.

Received for publication March 4, 2005. Accepted for publication October 20, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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NURSA Molecule Pages Link:

Nuclear Receptors:   AR
Coregulators:   E6AP  |  AIB1
Ligands:   R1881



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